CN114957415B - Streptavidin mutant and application and product thereof, gene, recombinant plasmid and genetically engineered bacterium - Google Patents

Streptavidin mutant and application and product thereof, gene, recombinant plasmid and genetically engineered bacterium Download PDF

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CN114957415B
CN114957415B CN202210498265.3A CN202210498265A CN114957415B CN 114957415 B CN114957415 B CN 114957415B CN 202210498265 A CN202210498265 A CN 202210498265A CN 114957415 B CN114957415 B CN 114957415B
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CN114957415A (en
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刁含文
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Nanjing Jujiang Biotechnology Co ltd
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Abstract

The invention provides a streptavidin mutant and application and products thereof, genes, recombinant plasmids and genetically engineered bacteria thereof, and relates to the technical field of biology. The streptavidin mutant provided by the invention truncates the amino acid sequence of the C end part of the wild streptavidin, selects 1-131 amino acids as a synthetic sequence, and introduces mutation points at two positions of Y44F and S46C. The affinity of the streptavidin mutant to biotin is obviously improved; the stability is good, the hydrolysis resistance to protease is obviously enhanced, and the technology development using the protease as a raw material is facilitated.

Description

Streptavidin mutant and application and product thereof, gene, recombinant plasmid and genetically engineered bacterium
Technical Field
The invention relates to the field of biotechnology, in particular to a streptavidin mutant and application and products, genes, recombinant plasmids and genetically engineered bacteria thereof.
Background
Streptavidin (hereinafter referred to as SA) is a protein having similar biological properties to avidin (hereinafter referred to as AV), and in 1963, chaiet was first discovered and reported when screening for anti-leptin.
Streptavidin can be specifically bound with 4 molecules of Biotin (Biotin) with a binding constant of 1×10 -15 and/M. Meanwhile, SA has no glycosyl and low isoelectric point, and has lower background than AV in detection, so that the detection sensitivity is greatly improved. The strepavidin-biotin system can be coupled to fluorescent substances such as antigens, antibodies, enzymes, oligonucleotide molecules and PE, so that strepavidin-biotin is widely used in biological reaction detection systems for detecting antigens, antibodies and nucleic acid molecules. At present, most of various detection kits based on the principles of enzyme-linked immune reaction, fluorescent molecular marker immune reaction and molecular hybridization at home and abroad adopt an SA-biotin system. At present, SA used in China is an expensive imported product, so that research and production of domestic SA have important practical significance and wide application prospect. The streptavidin and biotin-forming streptavidin-biotin amplifying system is a biological reaction amplifying system developed in the late seventies of the twentieth century, realizes signal amplification through specific combination between one molecule of avidin and four molecules of biotin, and can realize signal multistage amplification through multistage cascade amplification, so that the system has wider application in biology, particularly immunodetection.
Streptavidin is a protein with similar properties to avidin, and has the capacity of specifically binding to biotin, and the binding constant (Ka value) is as high as 10 15 M. Meanwhile, since streptavidin has lower isoelectric point than avidin and does not contain glycosyl chains, the sensitivity and specificity in detection are higher than that of avidin, and the application of a biotin-streptavidin system based on the streptavidin is more advantageous than that of a biotin-avidin system.
However, the coupling between the quaternary structure, stability and function of streptavidin makes it difficult to design stable, high affinity monomers for biotechnology applications. For example, the binding pocket of streptavidin tetramer is composed of residues from multiple subunits that cannot replicate in monomeric proteins.
In view of this, the present invention has been made.
Disclosure of Invention
It is a first object of the present invention to provide a streptavidin mutant to solve at least one of the above problems.
The second object of the invention is to provide the application of the streptavidin mutant in preparing detection products.
A third object of the present invention is to provide an inspection product.
The fourth object of the present invention is to provide a gene capable of encoding the aforementioned streptavidin mutant.
A fifth object of the present invention is to provide a recombinant plasmid.
The sixth object of the present invention is to provide a genetically engineered bacterium.
In a first aspect, the invention provides a streptavidin mutant, wherein the amino acid sequence of the streptavidin mutant is shown as SEQ ID NO. 1.
In a second aspect, the invention provides the use of a streptavidin mutant as described above in the preparation of a test product;
the detection products include protein detection products and nucleic acid detection products.
As a further technical scheme, the protein detection products comprise antigen detection products and antibody detection products.
In a third aspect, the invention provides an assay product comprising a streptavidin mutant as described above.
In a fourth aspect, the present invention provides a gene encoding the above-described streptavidin mutant.
In a fifth aspect, the present invention provides a recombinant plasmid comprising a vector and the above gene.
As a further technical scheme, the vector comprises pET-32a (+) plasmid.
In a sixth aspect, the present invention provides a genetically engineered bacterium, the genetically engineered bacterium containing the recombinant plasmid.
As a further technical scheme, the genetically engineered bacteria comprise escherichia coli.
Compared with the prior art, the invention has the following beneficial effects:
the streptavidin mutant provided by the invention truncates the amino acid sequence of the C end part of the wild streptavidin, selects 1-131 amino acids as a synthetic sequence, and introduces mutation points at two positions of Y44F and S46C. The affinity of the streptavidin mutant to biotin is obviously improved; the stability is good, the hydrolysis resistance to protease is obviously enhanced, and the technology development using the protease as a raw material is facilitated.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the expression results of m2-SA1 mutants;
FIG. 2 shows the purification results of the m2-SA1 mutant;
FIG. 3 is a comparison of the tolerance of m2-SA1 mutants to different proteases.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to embodiments and examples, but it will be understood by those skilled in the art that the following embodiments and examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The specific conditions are not specified, and the process is carried out according to conventional conditions or conditions suggested by manufacturers. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In a first aspect, the invention provides a streptavidin mutant, wherein the amino acid sequence of the streptavidin mutant is shown in SEQ ID NO. 1:
mdpskdskaqvsaaeagitgtwynqlgstfivtagadgaltgtfecavgnaesryvltgrydsapatdgsgtalgwtvawknnyrnahsattwsgqyvggaearintqwlltsgtteanawkstlvghdtft(SEQ ID NO.1)。
according to the invention, the amino acid sequence of the truncated C-end part of the wild streptavidin is selected from 1-131 amino acids as a synthetic sequence, and mutation points are introduced at two positions of Y44F and S46C to obtain the streptavidin mutant. The affinity of the streptavidin mutant to biotin is obviously improved; the stability is good, the hydrolysis resistance to protease is obviously enhanced, and the technology development using the protease as a raw material is facilitated. Wherein the amino acid sequence of the wild streptavidin is shown as SEQ ID NO. 3:
mdpskdskaqvsaaeagitgtwynqlgstfivtagadgaltgtyesavgnaesryvltgrydsapatdgsgtalgwtvawknnyrnahsattwsgqyvggaearintqwlltsgtteanawkstlvghdtftkvkpsaasidaakkagvnngnpldavqq(SEQ ID NO.3)。
the nucleotide sequence of streptavidin is shown as SEQ ID NO. 2:
ATGGACCCTTCTAAAGACTCCAAGGCTCAAGTTTCAGCCGCAGAAGCGGGTATTACTGGCACCTGGTATAATCAGTTAGGATCGACATTTATCGTCACGGCTGGGGCCGATGGTGCATTGACTGGCACCTACGAGAGTGCGGTAGGAAACGCTGAAAGTCGTTACGTGCTTACAGGGCGCTATGACAGCGCCCCCGCAACGGATGGTTCTGGCACTGCGCTCGGATGGACCGTTGCTTGGAAAAATAACTACCGAAATGCCCATTCCGCAACAACGTGGTCAGGGCAATATGTCGGTGGCGCGGAGGCTCGGATAAACACTCAGTGGCTACTGACCTCGGGAACAACGGAAGCCAATGCATGGAAGAGTACTTTAGTAGGGCACGACACCTTTACATAA(SEQ ID NO.2)。
in a second aspect, the invention provides the use of a streptavidin mutant as described above in the preparation of a test product. The detection products comprise protein detection products and nucleic acid detection products, wherein the protein detection products comprise antigen detection products and antibody detection products.
The strepitavidin-biotin system can be coupled with fluorescent substances such as antigens, antibodies, enzymes, oligonucleotide molecules, PE and the like, and is widely used in biological reaction detection systems for detecting antigens, antibodies and nucleic acid molecules. At present, most of various detection kits based on the principles of enzyme-linked immune reaction, fluorescent molecular marker immune reaction and molecular hybridization at home and abroad adopt an SA-biotin system. The streptavidin mutant provided by the invention has high biotin affinity and good stability, has the potential of replacing streptavidin, and can be used as a raw material for preparing detection products.
In a third aspect, the invention provides an assay product comprising a streptavidin mutant as described above. The detection product has high detection sensitivity and good stability.
In a fourth aspect, the present invention provides a gene encoding the above-described streptavidin mutant. For example, the gene may have the nucleotide sequence shown in SEQ ID NO. 2.
In a fifth aspect, the present invention provides a recombinant plasmid comprising a vector and the above gene. Among them, vectors include, but are not limited to, pET-32a (+) plasmids.
In a sixth aspect, the present invention provides a genetically engineered bacterium, the genetically engineered bacterium containing the recombinant plasmid. The genetically engineered bacterium may be, for example, escherichia coli.
The invention is further illustrated by the following specific examples and comparative examples, however, it should be understood that these examples are for the purpose of illustration only in greater detail and should not be construed as limiting the invention in any way.
The primers used in the following examples are shown in the following table:
EXAMPLE 1 preparation of streptavidin mutant
1) Optimizing the obtained mutant amino acid sequence by using UpGene, and obtaining a nucleic acid sequence SEQ ID NO.2 after optimizing;
2) After adding an N-terminal ATG start codon and a C-terminal TTA stop codon to SEQ ID NO.2, pMD18T-SA1 (PMD-18T with SEQ ID NO.2 inserted) was obtained by artificial synthesis;
3) Transferring the pMD18T-SA1/DH5a strain to LB for culture overnight, and taking 1ml of bacterial liquid to extract a plasmid DNA template;
4) Designed according to SEQ ID NO.2The sequence of the Y44F site-directed mutagenesis primer Y44F-F and Y44F-R is as follows, the pMD18T-SA1 plasmid extracted in the above step is used as a template, and MutUFO is used TM Fast Mutagenesis Kit quickly constructing and obtaining a Y44F mutant;
5) Transforming the mutant into DH5 alpha competent cells, and culturing the cells in a 37 ℃ incubator for 12 hours;
6) Performing PCR verification on single colonies of the transformation plate by using M13F and M13R as primers, and sending positive clones which are verified to be successful to sequencing;
7) The sequenced pMD18T-m1-SA1 recombinant plasmid is subjected to the mutation of a second point S46C, primers S46C-F and S46C-R are designed, the pMD18T-m1-SA1 recombinant plasmid is used as a template, and MutUFO is used TM Fast Mutagenesis Kit rapid construction to obtain S46C mutant;
8) Transforming the mutant into DH5 alpha competent cells, and culturing the cells in a 37 ℃ incubator for 12 hours;
9) Performing PCR verification on single colonies of the transformation plate by using M13F and M13R as primers, and sending positive clones which are verified to be successful to sequencing;
10 Sequencing and verifying to obtain a Y44F and S46C double mutant SA protein recombinant plasmid pMD18T-m2-SA1, and carrying out double enzyme digestion on the pMD18T-m2-SA1 through BamH I and Hind III to obtain a Y44F and S46C double mutant SA gene fragment;
11 Double enzyme digestion linearization is carried out on the pET32a (+) plasmid through BamH I and HindIII, and high-purity linear SA mutant gene fragments and pET32a (+) plasmid linear fragments are obtained through agarose gel electrophoresis;
12 The above gene fragment and vector fragment were ligated overnight at 16℃with M101 kit T4 DNALigase from Nanjing giant biotechnology Co.
13 Transferring the above-mentioned ligation product into BL21 competence, coating on ampicillin resistance plate, screening positive clone;
14 Colony PCR verification is carried out by using T7 and T7 terminator primers, and the pET32-m2-SA/BL21 strain which is successfully verified is transferred into 3ml LB culture medium for 8 hours;
15 Transferring to 500ml LB culture medium, culturing until OD600 = 0.6-0.8, adding 0.2mM IPTG, and inducing at 25deg.C for 12 hr;
16 The fermentation broth obtained by the above fermentation was collected, and the cells were resuspended in 20mM Tris-HCl, 500mM NaCl, and suspension Buffer pH=8.0, followed by disruption. Taking 1ml of suspension breaking centrifugation to collect cell breaking supernatant and cell breaking sediment, loading the cell breaking whole liquid together with SDS-PAGE, and running gel to observe the expression result, as shown in figure 1;
17 Further purifying the crude enzyme solution, co-precipitating by saturated ammonium sulfate (added to 75%), carrying out Ni column affinity chromatography, sephadex G50 fractionation and desalination, H cation exchange, Q anion exchange, sephadex G-200 desalination and separation, and ultrafiltration concentration to obtain high-purity m2-SA1 protein, wherein the result of purifying the protein is shown in figure 2.
Example 2 mutant SA affinity assay for biotin
The m2-SA1 mutant protein and the wild-type streptavidin obtained by the purification are taken and analyzed for affinity with biotin by a Biacore X100 biosensor. The test gave the following results:
EXAMPLE 3 hydrolysis of wild-type SA and m2-SA1 mutants by different proteases
As shown in the following table, the mutants were tested for their resistance to different proteases by selecting trypsin (trypsin), proteinase K (PRK), pepsin (pepsin), thermolysin (thermolysin) at 1% concentration for hydrolysis of 3. Mu.g of the above-mentioned m2-SA1 protein or SA at 37℃for 1h, and samples after treatment were subjected to SDS-PAGE, as shown in FIG. 3 (CK in the figure represents a blank, i.e.only m2-SA1 protein or SA was added).
In FIG. 3, SA or m2-SA1 and protease additions in CK, trypsin, PRK, pepsin and thermolysin groups are shown in the following table.
Note that: protease means: trypsin (trypsin), proteinase K (PRK), pepsin (pepsin), thermolysin (thermolysin), are added by "+" and not "-" are used.
As can be seen from FIG. 3, the m2-SA1 provided by the invention has stronger protease tolerance than the wild-type SA.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
SEQUENCE LISTING
<110> Nanjing great craftsman biotechnology Co., ltd
<120> streptavidin mutant and application and product thereof, gene, recombinant plasmid and genetically engineered bacterium
<160> 11
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Met Asp Pro Ser Lys Asp Ser Lys Ala Gln Val Ser Ala Ala Glu Ala
1 5 10 15
Gly Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser Thr Phe Ile Val
20 25 30
Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Phe Glu Cys Ala Val
35 40 45
Gly Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly Arg Tyr Asp Ser Ala
50 55 60
Pro Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp Thr Val Ala Trp
65 70 75 80
Lys Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr Trp Ser Gly Gln
85 90 95
Tyr Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp Leu Leu Thr
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Ser Gly Thr Thr Glu Ala Asn Ala Trp Lys Ser Thr Leu Val Gly His
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Asp Thr Phe Thr
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atggaccctt ctaaagactc caaggctcaa gtttcagccg cagaagcggg tattactggc 60
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actggcacct acgagagtgc ggtaggaaac gctgaaagtc gttacgtgct tacagggcgc 180
tatgacagcg cccccgcaac ggatggttct ggcactgcgc tcggatggac cgttgcttgg 240
aaaaataact accgaaatgc ccattccgca acaacgtggt cagggcaata tgtcggtggc 300
gcggaggctc ggataaacac tcagtggcta ctgacctcgg gaacaacgga agccaatgca 360
tggaagagta ctttagtagg gcacgacacc tttacataa 399
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Met Asp Pro Ser Lys Asp Ser Lys Ala Gln Val Ser Ala Ala Glu Ala
1 5 10 15
Gly Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser Thr Phe Ile Val
20 25 30
Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr Glu Ser Ala Val
35 40 45
Gly Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly Arg Tyr Asp Ser Ala
50 55 60
Pro Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp Thr Val Ala Trp
65 70 75 80
Lys Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr Trp Ser Gly Gln
85 90 95
Tyr Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp Leu Leu Thr
100 105 110
Ser Gly Thr Thr Glu Ala Asn Ala Trp Lys Ser Thr Leu Val Gly His
115 120 125
Asp Thr Phe Thr Lys Val Lys Pro Ser Ala Ala Ser Ile Asp Ala Ala
130 135 140
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Claims (8)

1. The streptavidin mutant is characterized in that the amino acid sequence of the streptavidin mutant is shown as SEQ ID NO. 1.
2. Use of a streptavidin mutant according to claim 1 for the preparation of a product for detecting biotin.
3. An assay product comprising a streptavidin mutant of claim 1.
4. A gene encoding the streptavidin mutant of claim 1.
5. A recombinant plasmid comprising a vector and the gene of claim 4.
6. The recombinant plasmid of claim 5, wherein the vector comprises a pET-32a (+) plasmid.
7. A genetically engineered bacterium comprising the recombinant plasmid of claim 5 or 6.
8. The genetically engineered bacterium of claim 7, wherein the genetically engineered bacterium comprises escherichia coli.
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Publication number Priority date Publication date Assignee Title
EP2493913A1 (en) * 2009-10-30 2012-09-05 Isis Innovation Limited High stability streptavidin mutant proteins
CN102731657A (en) * 2012-04-12 2012-10-17 海狸（广州）生物科技有限公司 Functional protein directional fixing method employing SLP
CN108752439A (en) * 2014-02-18 2018-11-06 赛威德医疗公司 Biotin variant, Streptavidin mutant and their application
CN114957415A (en) * 2022-05-09 2022-08-30 南京巨匠生物科技有限公司 Streptavidin mutant and application thereof, product, gene, recombinant plasmid and genetically engineered bacterium

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2493913A1 (en) * 2009-10-30 2012-09-05 Isis Innovation Limited High stability streptavidin mutant proteins
CN102731657A (en) * 2012-04-12 2012-10-17 海狸（广州）生物科技有限公司 Functional protein directional fixing method employing SLP
CN108752439A (en) * 2014-02-18 2018-11-06 赛威德医疗公司 Biotin variant, Streptavidin mutant and their application
CN114957415A (en) * 2022-05-09 2022-08-30 南京巨匠生物科技有限公司 Streptavidin mutant and application thereof, product, gene, recombinant plasmid and genetically engineered bacterium

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Atomic resolution structure of biotin-free Tyr43Phe streptavidin: what is in the binding site?;Freitag, S. 等;Acta Crystallogr D Biol Crystallogr;第55卷;1118-1126 *
The role of streptavidin and its variants in catalysis by biotinylated secondary amines;Nodling A.R. 等;Org Biomol Chem;第19卷(第47期);10424-10431 *
单价链霉亲和素的晶体结构解析;张敏;亚太传统医药(第9期);186-189 *
链霉亲和素突变体凝胶柱的制备及生物素修饰蛋白纯化;陈园坤;伦新新;李昂;王鹏举;张仪婷;赵晶;王辉;阎博;杨安钢;;细胞与分子免疫学杂志(04);304-309 *

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